The present invention is concerned with a new synthesis procedure for the preparation of novel carrier ampholyte mixtures suitable for isoelectric focusing of high molecular weight amphoteric substances (e.g., proteins).
During the process of stationary electrolysis, carrier ampholytes generate a uniform pH-gradient between the anode and the cathode. Preferentially, such carrier ampholyte mixtures consist of multiple chemical substances which differ from each other by the nature and the number of basic and acidic groups and, therefore, each ampholyte species has its own isoelectric point. In a particularly suitable system of carrier ampholytes, the isoelectric points of the different ampholyte species will cover the p-range 3-10 because most of the naturally occurring proteins are isoelectric within that pH-range.
Substantially uniform distribution of the isoelectric points of the various ampholyte species throughout the desired pH-range is an important factor for the formation of uniformly and linearly developed pH-gradients.
U.S. Pat. No. 3,485,736 describes a procedure for the synthesis of a mixture of polyamino-polycarboxylic acids suitable as carrier ampholytes for isoelectric focusing. This procedure utilizes a polyethylene-polyamine to which an, .alpha.,.beta.-unsaturated acid, e.g., acrylic acid, is chemically linked by an addition reaction. The simplest ampholyte formed by that reaction is a polyethylene-polyamine molecule to which one carboxylic acid molecule is linked forming a .beta.-aminocarboxylic acid group. Thereafter, .beta.-aminopolycarboxylic acids are obtained containing an increasing number of carboxylic acid groups in the molecule. The method of synthesis makes use of a known procedure of organic chemistry. To the aqueous solution of the polyethylene-polyamine a certain empirically determined amount of the aqueous solution of the carboxylic acid is added with simultaneous heating and stirring.
Under these conditions, obviously some of the ampholyte species which are more susceptible to the chemical reaction are obtained in higher yield whereas others will be formed in relatively low amounts. A suitable carrier ampholyte mixture forming a pH-gradient, however, should exhibit a uniform distribution of buffering capacity and conductivity throughout the pH-gradient. This is only achieved when the different carrier ampholyte species are contained in the mixture in about the same concentrations. Therefore, at the end of the chemical reaction, a fractionation of the carrier ampholyte mixture in an expansive multicompartment electrolysis equipment is necessary. A useful carrier ampholyte mixture is obtained by mixing appropriate amounts of materials from different electrolysis compartments.
Since the addition reaction between the unsaturated carboxylic acid and the polyethylene-polyamine exhibits very slow reaction kinetics at room temperature, the reaction is performed at elevated temperature. This, however, leads to undesired side products which cause coloration of the end product. Additionally, even at elevated temperature, the reaction knetics are rather slow and it is difficult to cause all of the unsaturated carboxylic acid to react. Such reaction, however, is essential to avoid artefacts during the isoelectric focusing procedure which may be caused by reaction of the unreacted unsaturated carboxylic acid with functional groups of proteins. For all these reasons, the known procedure is not completely satisfactory, and the art has searched for improvements.